Pharmaceutical compositions comprising macrolide diastereomers, methods of their synthesis and therapeutic uses

ABSTRACT

The disclosure relates to compositions comprising diastereomer of a macrolide exhibiting improved therapeutic profile in the context of inhibiting cell proliferation compared to the corresponding compositions comprising mixture of diastereomers. The disclosure further provides drug-ligand conjugates formed using diastereomer of the macrolide. The disclosure also provides novel method of preparation of diastereomer of the macrolide and their therapeutic uses.

Proliferative diseases are characterized by uncontrolled growth andspread of abnormal cells. If the spread of these cells is notcontrolled, it can result in death. Proliferative diseases, for examplecancer, can be treated by surgery, radiation, chemotherapy,hormone-based therapy and/or immunotherapy. A number of thesetreatments, particularly the chemotherapy, are based on the use ofanti-proliferative drugs which limit the spread of the abnormal cells.Anti-proliferative drugs, however, typically are indiscriminate in theirability to kill cells, affecting both normal and abnormal cells basedsimply on whether a cell is replicating. Regardless, mostanti-proliferative drugs require a relatively high concentration at thelocale of the abnormal cell proliferation to be effective. It is thiscombination of providing sufficient anti-proliferative drug at a site ofabnormal cell growth without also causing significant death to normalcells systemically or in the vicinity of these cells that thisdisclosure addresses.

Various approaches to targeted drug delivery have been tried, includingthe use of conjugates of tumor targeted probes (such as antibodies orgrowth factors) with toxins such as pseudomonas or diphtheria toxins.Conjugates for use in the treatment of cancer thereby target theanti-proliferative drug to a population of abnormal cells. Recently,conjugates that include the toxin maytansine have been employed for thetreatment of cancer. Maytansine has shown great effectiveness as ananti-proliferative agent but the compound's toxicity has provenproblematic toward normal cells. A need exists for developing maytansinebased conjugates that have sufficient activity for use as cancertherapeutics. The more active or effective the maytansine basedconjugate is at inhibiting or killing a population of abnormal cells thelower the concentration of the conjugate is required, the benefit beinga reduced overall risk of damaging normal cells.

Many anti-proliferative compounds exhibit asymmetric structures, such asthe Maytansinoid family of macrolides, and may therefore exist in theform of a racemic mixture, in the form of separate enantiomers withconfiguration “R” and “S”, or (+) and (−), per stereogenic center, andvarious diastereomers. The present disclosure shows that targeteddelivery of a single maytansinoid diastereomer exhibits improvedinhibitory cell proliferation profile as compared to delivery of itsrespective mixture of diastereomers. Therefore, diastereomers inaccordance with embodiments described herein can be used to preparemedicinal products with improved therapeutic profile that can be usefulfor the treatment of specific diseases and conditions, particularlycancer.

The present disclosure relates to compositions comprising a plurality ofdrug molecules for formula (I):

-   -   wherein:    -   X is

-   -   Y is Y₁ or Y₂ further wherein Y₁ is

or H;

-   -   Y₂ is —Cl, —Br, —I, or

-   -   Z is H or SO₃H;    -   R₁ and R₂ are independently selected from H or alkyl;    -   each n is independently 0 or an integer from 1 to 50;    -   and wherein the drug molecules present in the composition        comprises a mixture of at least two diastereomers, a first        diastereomer and a second diastereomer, further wherein said        first diastereomer and second diastereomer are otherwise        identical, except that said first and second diastereomers have        different stereochemical configuration at a chiral carbon        represented by (*) in formula X, wherein said chiral carbon atom        is a carbon atom that is bound to a sulfur atom, and said first        or second diastereomer is present in a diastereomeric excess of        greater than 50%.

In one embodiment, the disclosure provides compositions comprising aplurality of drug molecules of formula I, wherein n is 1, and R₁ and R₂are each independently hydrogen.

In another embodiment, the composition comprising a plurality of drugmolecules of formula I are present in the composition in adiastereomeric excess of about 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more.

In certain embodiments, the drug molecules of formula I are present inthe composition in a diastereomeric excess of from about 90% to about100%, or from about 95% to about 100%, or from about 98% to about 100%.

In another embodiment, the composition comprising a plurality of drugmolecules of formula I wherein one of the at least two diastereomers ischaracterized by a ¹H NMR spectra of FIG. 1 .

As used herein, the term “about”, when used in reference to a particularrecited numerical value, means that the value may vary from the recitedvalue by no more than 1%. For example, as used herein, the expression“about 100” includes 99 and 101 and all values in between (e.g., 99.1,99.2, 99.3, 99.4, etc.).

In another embodiment, the disclosure provides compositions comprising aplurality of drug molecules of formula I wherein the drug molecules arepresent in the composition in a diastereomeric excess of greater than50% and show greater anti-proliferative activity than a correspondingcomposition comprising drug molecules of formula I that are not presentin a diastereomeric excess of greater than 50%.

Further, the disclosure herein provides compositions comprising aplurality of drug molecules of formula I further comprising atherapeutically effective amount of a second or additional agentincluding, for example, a chemotherapeutic agent, an anti-inflammatoryagent, an antibiotic, and the like.

In an embodiment, the disclosure provides compositions comprising aplurality of drug molecules of formula I represented by the followingstructure:

or mixtures thereof in a diastereomeric excess of greater than 50%.

In one embodiment, one of the at least two diastereomers is a compoundof formula (i)

In another embodiment, one of the at least two diastereomers is acompound of formula (ii)

The present disclosure also provides a compound of formula (i) or (ii),

wherein the compound is stereomerically pure.

The present disclosure also provides compositions comprising a pluralityof ligand-drug conjugates of formula (II):

-   -   wherein:    -   A is

-   -   W is selected from S, O, or NR₃;    -   L is a ligand;    -   further wherein:    -   L is capable of binding to a cell or cell population;    -   R₁, R₂ and R₃ are each independently selected from H or alkyl;    -   n is 0 or an integer from 1 to 10;    -   p is an integer from 1 to 10;    -   and wherein the ligand-drug conjugates are present in the        composition in a diastereomeric excess of greater than 50%.

In one embodiment, the disclosure provides compositions comprising aplurality of ligand-drug conjugates of formula II, wherein n is 1, andR₁ and R₂ are each independently hydrogen.

In an embodiment, the compositions comprising a plurality of ligand-drugconjugates of formula II are present in the composition in adiastereomeric excess of about 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more. In certain embodiments, the drugmolecules of formula II are present in the composition in adiastereomeric excess of from about 90% to about 100%, or from about 95%to about 100%, or from about 98% to about 100%.

In another embodiment, the disclosure provides compositions comprising aplurality of ligand-drug conjugates of formula II in the compositions ina diastereomeric excess of greater than 50% and show greaterantiproliferative activity than a corresponding composition comprisingligand-drug conjugates of formula II that are not present in adiastereomeric excess of more than 50%.

In an embodiment, the disclosure provides compositions comprising aplurality of ligand-drug conjugates of formula II, wherein the ligand isan antibody or antigen-binding fragment thereof. The antibody orantigen-binding fragment thereof can be developed to specifically bind atumor-associated antigen. With respect to formula II, the n can be 1,and R₁ and R₂ can each be independently a hydrogen.

In certain embodiments, the disclosure provides compositions comprisinga plurality of ligand-drug conjugates of formula II, wherein the ligandis an antibody, or antigen-binding fragment thereof which specificallybinds a tumor-associated antigen, and the ligand-drug conjugates arepresent in the composition in a diastereomeric excess of more than about50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore.

In certain embodiments, the disclosure provides compositions comprisinga plurality of ligand-drug conjugates of formula II, wherein theantibody, or antigen-binding fragment thereof specifically binds atumor-associated antigen and further wherein the tumor-associatedantigen is selected from the group consisting of AFP, ALK, BAGEproteins, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX,caspase-8, CD20, CD40, CDK4, CEA, CLEC12A, cMET, CTLA4, cyclin-B1,CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EphA2,Fra-1, FOLR1, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH,glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT,IGF1R, LGR5, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and-12), MART-1, mesothelin, ML-IAP, Muc1, Muc16 (CA-125), MUM1, NA17,NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5,PCTA-1, PDGFR-α, PDGFR-β, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PLAC1, PRLR,PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3,Steap-1, Steap-2, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TNFRSF17, TRP-1,TRP-2, tyrosinase, and uroplakin-3.

In some embodiments, the antibody, or antigen-binding fragment haveamino acid sequences that vary from the above-mentioned antibodies butthat retain the ability to bind a given tumor-associated antigen. Suchvariant antibodies and antibody fragments can comprise one or moreadditions, deletions, or substitutions of amino acids when compared toparent sequence, but exhibit biological activity that is essentiallyequivalent to that of the described antibodies.

Two conjugates are considered bioequivalent if, for example, they arepharmaceutical equivalents or pharmaceutical alternatives whose rate andextent of absorption do not show a significant difference whenadministered at the same molar dose under similar experimentalconditions, either single dose or multiple dose. Some conjugates will beconsidered equivalents or pharmaceutical alternatives if they areequivalent in the extent of their absorption but not in their rate ofabsorption and yet may be considered bioequivalent because suchdifferences in the rate of absorption are intentional and are reflectedin the labeling, are not essential to the attainment of effective bodydrug concentrations on, e.g., chronic use, and are considered medicallyinsignificant for the particular drug product studied.

In one embodiment, two conjugates are bioequivalent if there are noclinically meaningful differences in their safety, purity, and potency.

In one embodiment, two conjugates are bioequivalent if a patient can beswitched one or more times between the reference product and thebiological product without an expected increase in the risk of adverseeffects, including a clinically significant change in immunogenicity, ordiminished effectiveness, as compared to continued therapy without suchswitching.

In one embodiment, two conjugates are bioequivalent if they both act bya common mechanism or mechanisms of action for the condition orconditions of use, to the extent that such mechanisms are known.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the conjugate or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the conjugate (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of a conjugate.

The present disclosure also provides a novel method for preparation of acomposition comprising a plurality of drug molecules of formula (I):

-   -   wherein:    -   X is

-   -   Y is Y₁ or Y₂ further wherein    -   Y₁ is

or H;

-   -   Y₂ is —Cl, —Br, —I, or

-   -   R₁ and R₂ are independently selected from H or alkyl;    -   each n is independently 0 or an integer from 1 to 50;    -   and wherein the drug molecules present in the composition        comprises a mixture of at least two diastereomers, a first        diastereomer and a second diastereomer, further wherein said        first diastereomer and second diastereomer are otherwise        identical, except that said first and second diastereomers have        different stereochemical configuration at a chiral carbon        represented by (*) in formula X, wherein said chiral carbon atom        is a carbon atom that is bound to a sulfur atom, and said first        or second diastereomer is present in a diastereomeric excess of        greater than 50%, the method comprising:    -   (a) providing a mixture comprising        -   (i) a starting material which has a formula III:

-   -   (ii) a compound of formula IV:

-   -   Y₁ is

or H;

-   -   Y₂ is —Cl, —Br, —I, or

-   -   Z is H or SO₃H;    -   R₁ and R₂ are independently selected from H or alkyl; and    -   each n is independently 0 or an integer from 1 to 50;        -   (iii) an organic solvent,        -   (iv) water, and        -   (v) a solid substrate;    -   (b) allowing the mixture of step (a) to react until some of the        starting material is converted to the compound of formula I; and    -   (c) removing crude compound of formula I from the mixture of        step (b).

In certain embodiments, the disclosure provides a method for preparingcomposition comprising a plurality of drug molecules of formula I,wherein the method further comprises step (d), further wherein step (d)comprises purifying the compound of formula I obtained in step (c) asexplained above.

In certain embodiments, the disclosure provides a method for preparingcompositions comprising a plurality of drug molecules of formula I,wherein the solid substrate is selected from the group consisting ofsilica gel, celite, alumina, a zeolite, and crushed molecular sieves.Other like solid substrates may also be utilized as long as the solidsubstrate allows for proper positioning of the macrolide III to allowstereoselective addition of maleimide IV.

In certain embodiments, the disclosure provides a method for preparingcompositions comprising a plurality of drug molecules of formula I,wherein n is 1, and R₁ and R₂ are each independently hydrogen.

In certain embodiments, the disclosure provides a method for preparingcompositions comprising a plurality of drug molecules of formula I,wherein the organic solvent comprises a polar aprotic solvent such asDMF, DMA, HMPT, NMP, acetonitrile, dioxane, acetone, DMSO, THF, ethylacetate, methyl acetate, methylene chloride, propylene carbonate ormixtures thereof.

In certain embodiments, the disclosure provides a method for preparingcompositions comprising a plurality of drug molecules of formula I,wherein the polar aprotic solvent comprises acetonitrile.

In certain embodiments, the disclosure provides a method for preparingcompositions comprising a plurality of drug molecules of formula I,wherein the organic solvent and the water are present in ratio of fromabout 1:1 to about 4:1 or from about 1:1 to about 10:1.

In certain embodiments, the disclosure provides a method for preparingcompositions comprising a plurality of drug molecules of formula I,wherein the molar ratio of the starting material having formula III andthe compound of formula IV is from about 1:1 to about 1:3.

In certain embodiments, the disclosure provides a method for preparingcompositions comprising a plurality of drug molecules of formula I,further comprising combining the compound of formula I with an antibodyor antigen-binding fragment thereof to make an antibody-drug conjugate.

In certain embodiments, the disclosure provides a method for preparingcompositions comprising a plurality of drug molecules of formula I,wherein the compound of formula I is attached to the antibody orantigen-binding fragment via an S, O, or NR₃.

In an embodiment, the disclosure provides a method for preparing acomposition comprising a plurality of drug molecules of formula I,wherein the drug molecules of formula I are present in the compositionin a diastereomeric excess of about 60% 70%, 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more.

In an embodiment, the disclosure provides a method for preparingcompositions comprising a plurality of drug molecules of formula Irepresented by the following structure:

in a diastereoselective excess of greater than 50%. Although certainexemplary methods for preparing the compositions of the presentinvention are set forth in the working examples herein, other methodsare contemplated within the scope of the present invention such as,e.g., chromatographic separation of a racemic mixture or mixture ofdiastereomers (e.g., HPLC on normal, reverse, or chiral stationary phaseusing polar aprotic solvent mixtures, etc.).

The present disclosure also relates to compositions wherein a pluralityof the drug molecules of formula I, or the ligand-drug conjugates offormula II, are contained within the compositions in a therapeuticallyeffective amount, and further comprising a pharmaceutically acceptablediluent, carrier or excipient.

In an embodiment, the disclosure provides compositions comprising aplurality of drug molecules of formula II further comprising atherapeutically effective amount of a second chemotherapeutic agent.

In numerous embodiments, the compositions comprise a compound of formula(I) and/or (II) of the present disclosure which may be administered incombination with one or more additional compounds or therapies.Co-administration and combination therapy are not limited tosimultaneous administration, separately or together, but also includesequential administrations.

Combination therapy includes administration of a single pharmaceuticaldosage formulation which contains a composition comprising one or morecompounds of formula (I) and/or (II) and one or more other therapeuticagents; as well as administration of a composition comprising compoundof formula (I) and/or (II) of the present disclosure and one or moreadditional agent(s) in its own separate pharmaceutical dosageformulation. For example, a composition comprising a compound of formula(I) and/or (II) and, a cytotoxic agent, a chemotherapeutic agent, or agrowth inhibitory agent can be administered to the patient together in asingle dosage composition such as a combined formulation, or each agentcan be administered in a separate dosage formulation. Where separatedosage formulations are used, a composition comprising a compound offormula (I) and/or (II) and one or more additional agents can beadministered concurrently, or separately at staggered times, i.e.,sequentially.

Non-limiting examples of such additional therapeutic agents includecytokine inhibitors (e.g., an interleukin-1 (IL-1) inhibitor (such asrilonacept or anakinra, a small molecule IL-1 antagonist, or ananti-IL-1 antibody); IL-18 inhibitor (such as a small molecule IL-18antagonist or an anti-IL-18 antibody); IL-4 inhibitor (such as a smallmolecule IL-4 antagonist, an anti-IL-4 antibody or an anti-IL-4 receptorantibody); IL-6 inhibitor (such as a small molecule IL-6 antagonist, ananti-IL-6 antibody or an anti-IL-6 receptor antibody); aspirin; NSAIDs;steroids (e.g., prednisone, methotrexate, etc.); low dose cyclosporineA; tumor necrosis factor (TNF) or TNF receptor inhibitors (e.g., a smallmolecule TNF or TNFR antagonist or an anti-TNF or TNFR antibody); uricacid synthesis inhibitors (e.g., allopurinol); uric acid excretionpromoters (e.g., probenecid, sulfinpyrazone, benzbromarone, etc.); otherinflammatory inhibitors (e.g., inhibitors of caspase-1, p38, IKK1/2,CTLA-4Ig, etc.); and/or corticosteroids. The additional therapeuticagent(s) may be administered prior to, concurrent with, or after theadministration of the one or more compounds of formula (I) and/or (II)(for purposes of the present disclosure, such administration regimensare considered the administration of one or more compounds of formula(I) and/or (II) “in combination with” a therapeutic agent).

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; eflornithine; elliptinium acetate; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone;mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinicacid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g.,paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (TAXOTERE®; Aventis Antony, France); chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also included in this definition areanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY 117018, onapristone, and toremifene(Fareston); and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptablesalts, acids or derivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially a cancer celleither in vitro or in vivo. Examples of growth inhibitory agents includeagents that block cell cycle progression (at a place other than Sphase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), TAXOL®, and topo II inhibitors such as doxorubicin,epirubicin, daunorubicin, etoposide, and bleomycin. Those agents thatarrest G1 also spill over into S-phase arrest, for example, DNAalkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of an activeagent and a pharmaceutically acceptable carrier. The term“pharmaceutically acceptable” means approved by a regulatory agency ofthe United States Federal or State government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

In an embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Where necessary, thecomposition may also include a solubilizing agent and a local anestheticsuch as lidocaine to ease pain at the site of the injection. Where thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade water orsaline. Where the composition is administered by injection, an ampouleof sterile water for injection or saline can be provided so that theingredients may be mixed prior to administration.

The active agents of the disclosure can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of the active agent of the disclosure, which will beeffective in the treatment of a medical condition, can be determined bystandard clinical techniques based on the present description. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the condition, and should be decided according to thejudgment of the practitioner and each subject's circumstances. However,suitable dosage ranges for intravenous administration are generallyabout 0.5 to 20 milligrams of active compound per kilogram body weight.Suitable dosage ranges for intranasal administration are generally about0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

In one embodiment, the disclosure provides a method of treatment of amedical disorder in an individual suffering from the medical disordercomprising administering to the individual an effective amount of acomposition comprising a compound of formula (I) and/or (II), andfurther comprising administering sequentially or consecutively anadditional therapy or administering at least one additional therapeuticagent.

In another embodiment, the disclosure relates to a method of treating adisease sensitive to treatment with said method, said method comprisingparenterally administering to a patient in need thereof atherapeutically effective dose of the composition comprising a compoundof formula (I) and/or (II).

The present disclosure further includes the use of any of thecompositions comprising compounds of formula (I), formula (II), formula(i), formula (ii), or a combination thereof and/or pharmaceuticalformulations thereof in the manufacture of a medicament for thetreatment, prevention and/or amelioration of a medical disorder.

The present disclosure further includes the use of any of thecompositions comprising compounds of formula (I), formula (II), formula(i), formula (ii), or a combination thereof and/or pharmaceuticalformulations thereof in the manufacture of a medicament for thetreatment, prevention and/or amelioration of a tumor.

While the embodiments herein have predominantly been described inrelation to antiproliferative disorders such as cancer, it is envisionedthat the compositions herein can also be useful in the treatment of amedical disorder selected from autoimmune diseases and otherimmunological diseases and dysfunctions, inflammatory diseases,infectious diseases, neurodegenerative diseases, bone disorders, andcardiovascular diseases. Further, any disorder that can benefit from thetargeted delivery of a toxic substance to particular cells, cell types,tissues and/or organs is within the scope of the present invention.

Finally, embodiments herein may include compositions comprising mixturesof compounds as represented by formula (I) and formula (II).

The references to certain embodiments made in the following descriptionare considered illustrative only of the principles of the disclosure.Further, since numerous modifications and changes will readily beapparent to those skilled in the art, it is not intended to limit thedisclosure to the exact construction and process shown as describedherein. Accordingly, all suitable modifications and equivalents may beresorted to as falling within the scope of the disclosure and as definedby the claims that follow.

The words “comprise”, “comprising”, “include” and “including” when usedin this specification and in the following claims are intended tospecify the presence of the stated features, integers, components, orsteps, but they do not preclude the presence or addition of one or moreadditional features, integers, components, or steps thereof.

General terms used in any of the embodiments herein can be defined asfollows; however, the meaning stated should not be interpreted aslimiting the scope of the term per se.

The term “conjugate” as used herein refers to compound having a Ligand,linker and Biologically Active Molecule. Illustrative examples includecompounds of formula (II).

The term “spacer” as used herein refers to chemical building blocks ofthe linker used to spatially separate the Ligand from the BiologicallyActive Molecule and to allow for catabolism of the linker inside ofcells.

The term “macrolide” as used herein refers to any Biologically ActiveMolecule having a macrolide ring.

The symbol

denotes the points of attachment.

The term “alkyl” as used herein refers to a hydrocarbon radical having astraight or branched chain or combinations thereof. Alkyl radicals canbe a univalent, a bivalent or a cyclic radical. Examples of univalentalkyl radicals are methyl, ethyl, 1-propyl, 2-propyl, n-butyl, isobutyl,sec-butyl, t-butyl, pentyl, neopentyl, hexyl, isohexyl, and the like. Asa way of illustration examples of bivalent alkyl radicals include

Examples of cyclic alkyl radicals include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and the like. Typical alkyl radicals have fromone to ten carbon atoms, one to nine carbon atoms, one to eight carbonatoms, one to seven carbon atoms, one to six carbon atoms, one to fivecarbon atoms, one to four carbon atoms, one to three carbon atoms, oneto two carbon atoms or one carbon atom. Typical cycloalkyl are 3 to 7member monocyclic ring radicals.

The phrase “pharmaceutically acceptable salt” as used herein refers toboth organic and inorganic salts of the conjugate compounds describedherein, e.g., compounds of formula (I), and (II). The salts arepharmaceutically acceptable and include: sulfate, citrate, nitrate,phosphate, ascorbate, bromide, gluconate, benzoate, oxalate,pantothenate, and the like. Note that pharmaceutically acceptable saltsherein may include more than one charged atom in its structure as wellas one or more counter ion. Preparation of conjugate compounds herein aspharmaceutically acceptable salts is well known to one of skill in theart.

The term “ligand” as used herein means any molecule or compound thatspecifically or selectively interacts with and/or binds to a secondmolecule or compound. In certain embodiments, a ligand is an antibody orantigen-binding fragment thereof. Other examples of ligands suitable foruse in the context of the present invention include, e.g., aptamers,peptides that specifically interact with a particular antigen (e.g.,peptibodies), receptor molecules, and antigen-binding scaffolds (e.g.,DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptiderepeat proteins, and other scaffolds based on naturally occurring repeatproteins, etc., [see, e.g., Boersma and Pluckthun, 2011, Curr. Opin.Biotechnol. 22:849-857, and references cited therein]).

Antibodies exist as intact immunoglobulins, or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. For example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H) by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′₂ dimer intoan Fab′ monomer. The Fab′ monomer is essentially Fab with part of thehinge region. While various antibody fragments are defined in terms ofthe digestion of an intact antibody, one of skill will appreciate thatsuch fragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv (scFv) single variable domains(Dabs)) or those identified using display libraries such as phage, E.coli or yeast display libraries (see, for example, McCafferty et al.(1990) Nature 348:552-554).

Methods for preparing antibodies are known to the art. See, for example,Kohler & Milstein (1975) Nature 256:495-497; Harlow & Lane (1988)Antibodies: a Laboratory Manual, Cold Spring Harbor Lab., Cold SpringHarbor, N.Y.). Antibodies that are isolated from organisms other thanhumans, such as mice, rats, rabbits, cows, can be made more human-likethrough chimerization or humanization.

The term “human antibody” as used herein is intended to includeantibodies having variable and constant regions derived from humanimmunoglobulin sequences. The human mAbs of the invention may includeamino acid residues not encoded by human immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo), for example in the CDRs and in particularCDR3. However, the term “human antibody”, as used herein, is notintended to include mAbs in which CDR sequences derived from thegermline of another mammalian species have been grafted onto human FRsequences.

The term “therapeutically effective amount” as used herein refers to anamount that produces the desired effect for which it is administered.The exact amount will depend on the purpose of the treatment, and willbe ascertainable by one skilled in the art using known techniques (see,for example, Lloyd (1999) The Art, Science and Technology ofPharmaceutical Compounding).

The term “racemate” as used herein means an equimolar mixture of a pairof enantiomers. The term racemate also refers to a racemic mixture.

The term “enantiomer” as used herein refers to compounds whichnon-superimposable with the mirror images of each other. Enantiomers mayexist in either the (R) or (S) configuration.

The term “stereoselective synthesis” refers to a chemical reaction thatleads to formation of a single stereoisomer or an enantiomer-enrichedmixture of isomers from among two or more possible stereoisomers.

The term “diastereomeric excess” refers to the difference between themole fraction of the desired single diastereomer as compared to theremaining diastereomers in a composition. Diastereomeric excess iscalculated as follows:

(amount of single diastereomer)−(amount of other diastereomers)/1

-   -   For example, a composition that contains 90% of 1 and 10% of 2,        3, 4, or a mixture thereof has a diastereomeric excess of 80%        [(90-10)/1]. A composition that contains 95% of 1 and 5% of 2,        3, 4, or a mixture thereof has a diastereomeric excess of 90%        [(95-5)/1]. A composition that contains 99% of 1 and 1% of 2, 3,        4, or a mixture thereof has a diastereomeric excess of 98%        [(99-1)/1]. The diastereomeric excess can similarly be        calculated for any one of 1, 2, 3, or 4.

The term “stereomerically pure” as used herein refers to a compoundwherein the indicated stereoisomer is present to a greater extent thanother stereoisomers of that compound, e.g., the compound is present indiastereomeric excess. In some embodiments, the stereomerically purecompounds described herein comprise 80% or greater, 85% or greater, 90%or greater, 95% or greater, or 97% or greater by weight of onestereoisomer of the compound.

Conjugates in accordance with various embodiments described herein canbe prepared by any known method in the art. An illustrative protocol forproducing conjugates is provided in the Examples below.

In one embodiment, the conjugates can be prepared by i) reacting aLigand with drug molecules of formula (I) to form a conjugate of formula(II), and ii) purifying the conjugate.

In an alternative embodiment, the conjugates are prepared by reacting aLigand, linker and biologically active macrolide in a single reaction.Once the conjugates in accordance with the invention are prepared theycan be purified.

In one embodiment, the compositions comprising drug molecules of formula(I) and/or compositions comprising conjugate compounds of formula (II)described herein can be evaluated for their ability to suppressproliferation of various cancer cell lines in vitro. For example,compositions comprising drug molecules or formula (I) and/orcompositions comprising conjugate compounds of formula (II) can beapplied to in vitro plated cancer cells for a predetermined number ofdays and surviving cells measured in assays by known methods.

The above specification, examples and data provide a completedescription of the manufacture and use of the composition of theinvention. Since many embodiments of the invention can be made withoutdeparting from the spirit and scope of the invention, the inventionresides in the claims hereinafter appended.

All references cited herein and in the Examples that follow, includingpatents, published patent applications, and literature references, areexpressly incorporated by reference in their entireties.

The description and Examples presented infra are provided to illustratethe subject invention. One of skill in the art will recognize theseExamples are provided by way of illustration only and are not includedfor the purpose of limiting the invention.

Embodiments disclosed herein are illustrated in greater detail by meansof the non-limiting examples described below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an ¹H-NMR spectrum ofMaytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxysuccinate(5). The ¹H-NMR spectrum is consistent with a single diastereomerpresent in at least 95% diastereomeric excess since the spectrum is notcomplicated by resonances attributable to the other diastereomer. (Forcomparative purposes Example 2 sets forth the ¹H-NMR spectrum of themixture of diastereomers).

FIG. 2 illustrates an ¹H-NMR spectrum of mixture of diastereomers ofMaytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxysuccinate(6).

FIG. 3 illustrates that in SKBR3 cells the single diastereomer compoundconjugate HER2-5 (in vitro and in vivo lots) possessed an IC50 value of0.3 nM versus 0.9 nM for the mixture of diastereomer conjugate HER2-6.

FIG. 4 illustrates that in BT474 cells the single diastereomer compoundconjugate HER2-5 (in vitro) in possessed an IC₅₀ value of 4.6 nM whilethe HER2-5 (in vivo) lot had an IC₅₀ value of 4.0 nM versus 11.6 nM forthe mixture of diastereomer conjugate HER2-6.

FIG. 5 illustrates that in NCI-N87 cells the single diastereomercompound conjugate HER2-5 (in vitro) possessed an IC₅₀ value of 0.6 nMwhile the HER2-5 (in vivo) lot had an IC₅₀ value of 0.4 nM versus 1.0 nMfor the mixture of diastereomer conjugate HER2-6.

FIG. 6 illustrates that in HEK293/hEGFRvIII cells the singlediastereomer compound conjugate EGFRvIII-5 possessed an IC₅₀ value of0.4 nM while the mixture of diastereomer conjugate EGFRvIII-6 had anIC₅₀ value of 0.5 nM.

FIG. 7 illustrates that in MMT/hEGFRvIII cells the single diastereomercompound conjugate EGFRvIII-5 possessed an IC₅₀ value of 0.5 nM whilethe mixture of diastereomer conjugate EGFRvIII-6 had an IC₅₀ valuealmost 20 fold higher at 9.8 nM.

FIG. 8 illustrates that in U251/hEGFRvIII cells the single diastereomercompound conjugate EGFRvIII-5 possessed an IC₅₀ value of 2.4 nM whilethe mixture of diastereomer conjugate EGFRvIII-6 had an IC₅₀ value of3.3 nM.

FIG. 9 illustrates that in Ovcar-3 cells the single diastereomercompound conjugate MUC16-5 possessed an IC₅₀ value of 6.3 nM while themixture of diastereomer conjugate MUC16-6 had an IC₅₀ value of 16.0 nM.

FIG. 10 illustrates that in PC3/MUC16 long cells the single diastereomercompound conjugate MUC16-5 in possessed an IC₅₀ value of 0.34 nM whilethe mixture of diastereomer conjugate MUC16-6 had an IC₅₀ value at 0.80nM.

FIG. 11 illustrates tumor growth curves in mice following dosing withHER2-5 and control reagents. Mice received PBS vehicle (▪), 300 ug/kgDM1-SMe (●) and Isotype Control mAb at 15 mg/kg (▾). Mice also receivedHER2 mAb (▴), HER2-5 (Δ) and Isotype Control-5 (∇) at doses of 1 mg/kg(A), 5 mg/kg (B) and 15 mg/kg (C). Mice received 3 once weekly doses ofconjugates and control agents as indicated by the black arrows (T.Groups are N=8, Mean±SE).

FIG. 12 illustrates change in tumor volume following dosing with HER2-5and control reagents at termination of the PBS vehicle control group onDay 79 post tumor implantation. Individual tumor sizes are shown foreach dosing group. Mice received PBS vehicle (▪), 300 ug/kg DM1-SMe (●)and Isotype Control mAb at 15 mg/kg (▾). Mice also received HER2 mAb(▴), HER2-5 (Δ) and Isotype Control-5 (∇) at doses of 1 mg/kg (A), 5mg/kg (B) and 15 mg/kg (C). Groups are N=8, Mean±SD.

FIG. 13 illustrates percentage change in animal weights following dosingwith HER2-5 and control reagents. Mice received PBS vehicle (▪), 300ug/kg DM1-SMe (●) and Isotype Control mAb at 15 mg/kg (▾). Mice alsoreceived HER2 mAb (▴), HER2-5 (Δ) and Isotype Control-5 (∇) at doses of1 mg/kg (A), 5 mg/kg (B) and 15 mg/kg (C). Mice received 3 once weeklydoses of conjugates and control agents as indicated by the black arrows(T. Groups are N=8, Mean±SE).

EXAMPLES Experimental Details

Proton NMR spectra (for compounds that could not be detected by UV) wereacquired on a Varian Inova 300 MHz instrument, while mass spectra werecollected on an Agilent 1100 series LC/MSD with electrospray ionizationsource and quadrupole ion trap analyzer. All conjugates were analyzedusing a Bruker ultraFleXtreme MALDI-TOF-TOF mass spectrometer. Allstarting materials and solvents were purchased commercially and usedwithout purification, unless otherwise noted.

Example 1

Synthesis of Maleimidylmethyl-4-trans-cyclohexylcarboxy-succinate (3):Maleimidylmethyl-4-trans-cyclohexanecarboxylic acid (2): The titlecompound was prepared in two steps (Step A and Step B) using modifiedversions of the methods described by Marnett et al. (J. Med. Chem.,1996, 39, 1692-1670).

Step A: A solution of trans-4-aminomethylcyclohexane carboxylic acid(7.00 g, 44.5 mmol) in 1,4-dioxane (70 mL) was treated with maleicanhydride (4.89 g, 49.9 mmol) and stirred at ambient temperature for 48h. The reaction was evaporated in vacuo to a white solid that can bestored or carried on to the next step without further purification. ¹HNMR (300 MHz, DMSO-d₆) δ 9.11 (m, 1H), 6.44 (d, 1H, J=13 Hz), 6.24 (d,1H, J=13 Hz), 3.05 (t, 2H, J=6 Hz), 2.13 (tt, 1H, J=12 Hz, 4 Hz), 1.90(m, 2H), 1.75 (m, 2H), 1.44 (m, 1H), 1.28 (m, 2H), 0.96 (m, 2H).

Step B: The maleamic acid from Step A (36.8 g, 144 mmol) and sodiumacetate (13.6 g, 165 mmol) were dissolved in acetic anhydride (368 mL),sealed in a glass reaction vessel, and heated to 120° C. for 3 hours.The cooled reaction mixture (a black syrup) was poured onto water (3 L),stirred, and extracted with dichloromethane. The organic layer was driedover Na₂SO₄, filtered over a sintered glass funnel, and the clearfiltrate evaporated and dried under high vacuum giving the titlecompound as a yellow solid (7.00 g, 20%). ¹H NMR (300 MHz, CDCl₃) δ 6.73(s, 2H), 3.40 (d, 2H, J=7 Hz), 2.28 (m, 1H), 2.06 (m, 2H), 1.75 (m, 3H),1.42 (m, 2H), 1.03 (m, 2H).

Maleimidylmethyl-4-trans-cyclohexanecarboxysuccinate (3): The product ofthe preceding step B (10.0 g, 42.1 mmol) was dissolved indichloromethane (50 mL) under Ar, treated with N-hydroxysuccinimide(7.27 g, 63.2 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (EDAC, 12.4 g, 64.5 mmol), and the reaction was stirred atambient temperature overnight. The resulting brown solution was dilutedwith dichloromethane, washed with water and brine, dried over Na₂SO₄,filtered over a sintered glass funnel, and the filtrate concentrated anddried in vacuo. This product was then dissolved in hot ethyl acetate,treated with activated charcoal (1.5 g), filtered, and the filtratecooled. Filtration of the crystalline product, washing with cold ethylacetate, and suction drying then gave the title compound as a tan solid(5.52 g, 39%). ¹H NMR (300 MHz, CDCl₃) δ 6.72 (s, 2H), 3.42 (m, 2H),2.85 (br s, 4H), 2.56 (tt, 1H, J=12 Hz, 4 Hz), 2.18 (m, 2H), 1.80 (m,2H), 1.70 (m, 1H), 1.56 (m, 2H), 1.09 (m, 2H).

Example 2

Maytansin-3-N-methyl-L-alanine-propanamide-3-thiol (4): The titlecompound, known in the art as DM1, was prepared using a modified versionof the method described by Whitesides et al. (J. Org. Chem., 1991, 56,2648-2650). Maytansin-3-N-methyl-L-Ala-(3-methyldisulfanyl) propanamide(DM1-SMe, 2.42 g, 3.09 mmol, prepared in a manner similar to Ho andCarrozzella, U.S. Pat. Appl. 2007/0037972 A1) was dissolved inacetonitrile (30 mL), treated with a solution oftris(2-carboxyethyl)phosphine hydrochloride (8.23 g, 28.7 mmol) in water(30 mL), the pH was raised to ca. 3 with the addition of saturatedaqueous NaHCO₃ (5 mL), the flask was purged with Ar, and the reactionwas stirred at ambient temperature under a rubber septum (vented due toeffervescence). After 2 h, the reaction was treated with brine (ca. 100mL), bubbled with Ar for 5 minutes (to remove the free methylmercaptan),and the phases separated. The aqueous phase was extracted twice withethyl acetate (EtOAc), saturated with NaCl, and extracted twice morewith EtOAc. The combined organic layers were then dried over Na₂SO₄,filtered, and the filtrate concentrated and dried in vacuo to give thetitle compound as a white solid (2.24 g, 98%). ¹H NMR (300 MHz,CDCl₃/CD₃OD) δ 6.76 (d, 1H, J=1.5 Hz), 6.63 (d, 1H, J=11 Hz), 6.59 (d,1H, J=1.5 Hz), 6.35 (m, 2H), 5.59 (dd, 1H, J=15 Hz, 9 Hz), 5.36 (q, 1H,J=6.5 Hz), 4.68 (dd, 1H, J=12 Hz, 3 Hz), 4.21 (t, 1H, J=10 Hz), 3.92 (s,3H), 3.60 (d, 1H, J=13 Hz), 3.42 (d, 1H, J=9 Hz), 3.29 (s, 3H), 3.14 (s,3H), 3.05 (d, 1H, J=13 Hz), 2.95 (d, 1H, J=10 Hz), 2.77 (s, 3H),2.75-2.47 (m, 6H), 2.11 (dd, 1H, J=14 Hz, 3 Hz), 1.58 (s, 3H), 1.47 (d,1H, J=14 Hz), 1.40 (m, 1H), 1.22 (m, 6H), 0.73 (s, 3H). MS (ESI, pos.):calc'd for C₃₅H₄₈ClN₃O₁₀S, 737.3; found 738.3 (M+H), 720.3 (M−H₂O+H).

Maytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxysuccinate(5): The following procedure describes a new method not known in theart. The product of the preceding step (4, 2.23 g, 3.02 mmol) andMaleimidylmethyl-4-cyclohexanecarboxysuccinate (3, 1.50 g, 4.49 mmol)were dissolved in 4:1 acetonitrile/water (75 mL), treated with finesilica gel scraped from a preparative TLC plate (11.2 g), the flaskpurged with Ar, and the mixture stirred at ambient temperature underrubber septum. After 18 hours, more 3 (0.77 g, 2.3 mmol) andacetonitrile (MeCN, 10 mL) were added, and the reaction stirred anadditional 6 hours. The mixture was filtered over Celite, solids washedwith MeCN and ethyl acetate (EtOAc), and the filtrate concentrated invacuo to a gold solid, which was purified by flash column chromatographyon an 120 g silica gel cartridge (50-100% 1:1 EtOAc/MeCN indichloromethane over 33 min, 75 mL/min). Evaporation and drying of thepure column fractions in vacuo gave the title compound as acream-colored solid (2.09 g). Concentration of the impure fractions andrepurification on an 80 g silica gel cartridge as above gave anadditional batch of cream-colored solid (0.22 g), and brought the totalyield of title compound to 2.31 g (71%). ¹H NMR (300 MHz, CDCl₃) δ 6.85(d, 1H, J=4 Hz), 6.72 (m, 1H), 6.65 (d, 1H, J=4 Hz), 6.44 (dd, 1H, J=15Hz, 11 Hz), 6.25 (s, 1H), 5.67 (dd, 1H, J=16 Hz, 9 Hz), 5.41 (m, 1H),4.79 (d, 1H, J=11 Hz), 4.30 (t, 1H, J=11 Hz), 3.72 (m, 2H), 3.51 (d, 1H,J=9 Hz), 3.37 (m, 4H), 3.27 (m, 1H), 3.23 (s, 3H), 3.16-2.99 (m, 4H),2.85 (m, 7H), 2.62 (m, 3H), 2.39 (ddd, 1H, J=19 Hz, 12 Hz, 4 Hz), 2.18(br m, 2H), 1.77 (br m, 3H), 1.66 (s, 3H), 1.60-1.47 (m, 4H), 1.31 (m,6H), 1.05 (m, 2H), 0.82 (s, 3H). MS (ESI, pos.): calc'd forC₅₁H₆₆ClN₅O₁₆S, 1071.4; found 1072.4 (M+H), 1054.9 (M−H₂O+H); [α]²⁰_(589nm)=−52.4 (c=0.00301, MeOH). See FIG. 1 for ¹H-NMR of singlediastereomer.

Example 3

Mixture of diastereomers ofMaytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxysuccinate(6): A sample of the mixed stereoisomers of 5 was synthesized accordingto US Patent Application 20100129314, Example XI. ¹H NMR (300 MHz,CDCl₃) δ 6.85-6.6 (m), 6.4 (m), 6.1 (m), 5.8-5.4 (m), 5.2 (m), 4.92-4.79(m), 4.4-4.1 (m), 4.03 (s), 3.82 (m), 3.8-2.2 (m), 2.1 (m), 2.07 (s),2.0-0.8 (m). MS (ESI, pos.): calc'd for C₅₁H₆₆ClN₅O₁₆S, 1071.4; found1072.4 (M+H). See FIG. 2 for ¹H-NMR of mixture of diastereomers.

Example 4

RacemicMaytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxylicacid (8): A solution oftrans-1,4-(maleimidomethyl)cyclohexane-1-carboxylic acid 7 (167 mg,0.701 mmol) in 1.2-dimethoxyethane (8 mL) was added to a solution of 4(340 mg, 0.461 mmol) in 1.2-dimethoxyethane (15 mL). The mixture wasthen treated with pH 7.5 buffer (20 mL) and a few drops of saturatedaqueous NaHCO₃ to maintain the pH. The reaction mixture was stirredovernight at room temperature under argon and then concentrated underreduced pressure. The crude residue was purified by reverse phasechromatography using a C18 column, 20-40 micron column (100 g), elutingwith a gradient (10 95% over 25 mins) of acetonitrile (0.1% AcOH) inwater (0.1% AcOH), and lyophilized to give 8 (330 mg, 0.338 mmol, 73%yield) as a white solid. MS m/z: 977.2 [MH+], 957.2 [M−18], 999.2[M+Na]; Purity: >98% (by LC/MS).

Example 5

Chiral Separation of 8 to Single Diastereomers(R*)-Maytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxylicacid (9) and(S*)-Maytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxylicacid (10) (S* and R* represent a single stereoisomer of unknownchirality): The diastereomeric mixture of compounds 8 (20 mg) wasdissolved in 0.5 ml of acetonitrile and separated using semi-prep Chiralcolumn (Chiralcel OJ, Solvent system, 6:1:1 Hexanes:IPA:Ethanol) toafford 3.5 mg of 10 as the faster-running compound, MS m/z: 977.2 [MH+],957.2 [M−18], 999.2 [M+Na]; Purity: >95% (by LC/MS), RT=32 min and 4.6mg of 9 as the slower-running compound, MS m/z: 977.2 [MH+], 957.2[M−18], 999.2 [M+Na]; Purity: >95% (by LC/MS), Rf=48 min.

Example 6

Synthesis of(S*)-Maytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxylicacid (11): A solution of 10 (2.5 mg, 0.0026 mmol) was dissolved indichloromethane (1 mL), then treated with N-hydroxysuccinimide (NHS, 6.0mg, 0.052 mmol) and 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimidehydrochloride (EDCI, 13 mg, 0.065 mmol). The reaction mixture wasstirred overnight at room temperature under argon, washed with waterfollowed by brine, dried over anhydrous sodium sulfate, and filtered.The solvent was evaporated under reduced pressure to give the cruderesidue, which was purified by reverse phase chromatography using a C18,20-40 micron column (30 g), eluting with a gradient (10-95% over 18 min)of acetonitrile (0.1% AcOH) in water (0.1% AcOH), and lyophilized toafford 11. MS m/z: 1073.2 [MH+], 1054.4[M−18]; Purity: 95% (by LC/MS).

Example 7

(R*)-Maytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxylicacid (12): A solution of 9 (3, mg, 0.003 mmol) was dissolved indichloromethane (1 mL), then treated with N-hydroxysuccinimide (NHS, 3.0mg, 0.026 mmol) and 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimidehydrochloride (EDCI, 7 mg, 0.036 mmol). The reaction mixture was stirredovernight at room temperature under argon, washed with water followed bybrine, dried over anhydrous sodium sulfate, and filtered. The solventwas evaporated under reduced pressure to give the crude residue, whichwas purified by reverse phase chromatography using C18 column, 20-40micron (15 g), eluting with a gradient (10-95% over 18 min) ofacetonitrile (0.1% AcOH) in water (0.1% AcOH), lyophilized to afford 12.MS m/z: 1073.2 [MH+], 1054.4[M−18]; Purity: 95% (by LC/MS).

Example 8

Conjugate Preparation and Characterization.

Two different maytansine-linker compositions prepared according to theprevious Examples (Compound 5 and Compound 6) were conjugated to variousanti-tumor antigen monoclonal antibodies. Compound 5 comprisespredominantly a single diastereomer of the linker-DM1 cytotoxiccompound, whereas Compound 6 comprises a mixture of various linker-DM1diastereomers. The antibodies used in this Example were an anti-HER2antibody having variable regions derived from humAb4D5-8 from Carter etal, PNAS 1992 89 4285, an anti-EGFRvIII antibody having variable regionsderived from clone 131 from WO2013075048 A1, and an anti-MUC16 antibodyhaving variable regions derived from clone 3A5 from WO2007001851.

The antibodies were expressed in CHO cells and purified by Protein A. Anon-binding isotype control antibody derived from an infectious diseaseantigen having no relation to oncology was also used in this Example.

The antibodies (10 mg/ml) in 50 mM HEPES, 150 mM NaCl, pH 8.0, and 10%(v/v) DMA were conjugated with a 6 fold excess of compound 5 or 6 for 1hour at ambient temperature. The conjugates were purified by sizeexclusion chromatography and sterile filtered. Protein and linkerpayload concentrations were determined by UV spectral analysis andMALDI-TOF mass spectrometry. Size-exclusion HPLC established that allconjugates used were >95% monomeric, and RP-HPLC established that therewas <0.5% unconjugated linker payload. Yields are reported in Table 1based on protein. All conjugated antibodies were analyzed by UV forlinker payload loading values according to Hamblett et al, Cancer Res2004 10 7063 and by mass difference, native versus conjugated. Theresults are summarized in Table 1.

TABLE 1 ε252 nm (cm⁻¹ M⁻¹) ε280 nm (cm⁻¹ M⁻¹) Compound 5  26790 5700 6*26790 5700 Antibody HER2 81847 215388 EGFRvIII 79579 209420 MUC16 88671248380 Isotype Control 81718 233480 Antibody Payload:AntibodyPayload:Antibody Conjugate (UV) (MS) Yield % HER2-5 (in vivo) 2.7 2.7 66HER2-5 (in vitro) 3.1 2.4 75 HER2-6 (in vitro) 2.9 2.4 70 EGFRvIII-5 2.82.3 56 EGFRvIII-6 2.9 2.2 56 MUC16-5 2.4 2.0 76 MUC16-6 2.3 2.1 96Isotype Control-5 3.3 3.3 67 *Extinction coefficients were used fromcompound 5

Example 9

In Vitro Cytotoxicity Assays.

Cells were seeded in PDL-coated 96 well plates at 10,000 (SK-BR-3 andNCI-N87), 15,000 (BT-474), 3000 (Ovcar-3 and PC3/Muc16), 2000(HEK293/hEGFRvIII), 1500 (U251/hEGFRvIII), or 400 (MMT/hEGFRvIII) cellsper well in complete growth media and grown overnight. For cellviability curves, serially diluted antibody-drug conjugates or freepayload were added to the cells at final concentrations ranging from 1μM to 1 pM and incubated for 3 days. Each concentration was run induplicate and reported with the respective standard deviation. Cellswere incubated with CCK8 (Dojindo) for the final 1-3 h and theabsorbance at 450 nm (OD₄₅₀) was determined on a Flexstation3 (MolecularDevices). Background OD₄₅₀ levels from digitonin (40 nM) treated cellswere subtracted from all wells and viability is expressed as apercentage of the untreated controls. IC₅₀ values were determined from afour-parameter logistic equation over a 10-point response curve(GraphPad Prism). All curves and IC₅₀ values are corrected for payloadequivalents based on the loading from the MALDI-TOF experiment.

In SKBR3 cells (breast cancer line), natively expressing HER2 at 607fold above isotype control binding, the single diastereomer compoundconjugate HER2-5 (in vitro and in vivo lots) possessed an IC₅₀ value of0.3 nM versus 0.9 nM for the mixture of diastereomer compound conjugateHER2-6 (Table 2, FIG. 3 ). A small in vitro lot was conjugated first andonly used for cell proliferation assays, while a larger in vivo lot wasthen conjugated and used for both in vitro and in vivo experiments. Thenaked HER2 antibody had little anti-proliferation activity.

In BT474 cells (breast cancer line), natively expressing HER2 at 426fold above isotype control binding, the single diastereomer compoundconjugate HER2-5 (in vitro) possessed an IC₅₀ value of 4.6 nM while theHER2-5 (in vivo) lot had an IC₅₀ value of 4.0 nM versus 11.6 nM for themixture of diastereomer compound conjugate HER2-6 (Table 2, FIG. 4 ).The naked HER2 antibody had little anti-proliferation activity.

In NCI-N87 cells (breast cancer line), natively expressing HER2 at 869fold above isotype control binding, the single diastereomer compoundconjugate HER2-5 (in vitro) possessed an IC₅₀ value of 0.6 nM while theHER2-5 (in vivo) lot had an IC₅₀ value of 0.4 nM versus 1.0 nM for themixture of diastereomer compound conjugate HER2-6 (Table 2, FIG. 5 ).The naked HER2 antibody had little anti-proliferation activity.

In HEK293/hEGFRvIII cells, expressing hEGFRvIII at 360 fold aboveisotype control binding, both conjugates (single and mixture ofdiastereomer) exhibited IC₅₀ values of 0.4 nM (Table 3, FIG. 6 ). Thenaked EGFRvIII antibody had little anti-proliferation activity.

In MMT/hEGFRvIII cells, expressing hEGFRvIII at 280 fold above isotypecontrol binding, the single diastereomer compound conjugate EGFRvIII-5possessed an IC₅₀ value of 0.5 nM while the mixture of diastereomercompound conjugate EGFRvIII-6 had an IC₅₀ value of 9.8 nM (Table 2, FIG.7 ). The naked EGFRvIII antibody had little anti-proliferation activity.

In U251/hEGFRvIII cells, expressing hEGFRvIII at 165 fold above isotypecontrol binding, the single diastereomer compound conjugate EGFRvIII-5possessed an IC₅₀ value of 2.4 nM while the mixture of diastereomercompound conjugate EGFRvIII-6 had an IC₅₀ value of 3.3 nM (Table 3, FIG.7 ). The naked EGFRvIII antibody had little anti-proliferation activity.

In Ovcar-3 cells (ovarian cancer line), natively expressing MUC16 at 373fold above isotype control binding, the single diastereomer compoundconjugate MUC16-5 possessed an IC₅₀ value of 6.3 nM while the mixture ofdiastereomer compound conjugate MUC16-6 had an IC₅₀ value of 16.0 nM(Table 4, FIG. 8 ). The naked Muc16 antibody had littleanti-proliferation activity.

In PC3/MUC16 cells, expressing MUC16 at 105 fold above isotype controlbinding, the single diastereomer compound conjugate MUC16-5 possessed anIC₅₀ value of 0.34 nM while the mixture of diastereomer compoundconjugate MUC16-6 had an IC₅₀ value of 0.8 nM (Table 4, FIG. 9 ). Thenaked Muc16 antibody had little anti-proliferation activity.

In FIGS. 3, 4, and 5 maytansin-3-N-methyl-L-Ala-(3-methyldisulfanyl)propanamide (DM1-SMe, prepared according to Ho and Carrozzella, U.S.Pat. Appl. 2007/0037972 A1) was chosen to represent the payload in theseassays. Compound 4 would be too reactive to use in vitro or in vivo,thereby giving unreliable results.

The in vitro results are summarized in Tables 2-4 on a target basisbelow. This Example demonstrates that anti-tumor antibody-drugconjugates comprising the single diastereomer drug of the presentinvention, in most cases, exhibited greater in vitro killing potencythan the corresponding antibody-drug conjugates comprising mixture ofdiastereomers. For the targets analyzed, the single diastereomerantibody-drug conjugates were typically on the order of 2- to 3-foldmore potent than the corresponding mixture conjugates, depending on theparticular cell lines tested.

TABLE 2 Antibody SKBR3 BT474 N87 Conjugate IC50 (nM) IC50 (nM) IC50 (nM)HER2-5 (in vivo) 0.3 4.0 0.4 HER2-5 (in vitro) 0.3 4.6 0.6 HER2-6 (invitro) 0.9 11.6 1.0

TABLE 3 Antibody HEK293/hEGFRvIII MMT/hEGFRvIII U251/hEGFRvIII ConjugateIC50 (nM) IC50 (nM) IC50 (nM) EGFRvIII-5 0.4 0.5 2.4 EGFRvIII-6 0.5 9.83.3

TABLE 4 Antibody Ovcar-3 PC3/MUC16 Conjugate IC50 (nM) IC50 (nM) MUC16-56.3 0.34 MUC16-6 16.0 0.80

Example 10

In Vivo Studies.

To determine the in vivo efficacy of the anti-HER2 single-diastereomerconjugate (“HER2-5”), studies were performed in mice bearing HER2+gastric cancer xenografts, as efficacy had been previously reported inthis model by Barok et al (Barok M et al, Can Letters 2011).Specifically, 5×10⁶ NCI-N87 cells (ATCC CRL-5822) were implantedsubcutaneously into the lower right flank of CB-17 SCID mice (Taconic).Once tumors had reached an average volume of 150 mm³, mice wererandomized in to groups of eight and dosed with HER2-5 or controlreagents. Control reagents included PBS vehicle, free DM1-SMe, isotypecontrol, isotype control-5, or HER2. Mice received once weekly doses forthree weeks and tumor volumes and body weights were monitored twiceweekly throughout the study. Conjugates were dosed at 1, 5 and 15 mg/kg,as these doses had been shown to be effective in previous in vivostudies by Lewis-Phillips et al (Lewis-Phillips G et al., Can Res 2008).

In the current N87 tumor model, HER2-5 demonstrated clear anti-tumorefficacy, with doses of 5 and 15 mg/kg leading to significant decreasesin initial tumor volume and eradication of some tumors at the higherdose (FIGS. 11 and 12 ). A significant delay in tumor growth relative tocontrol agents was also observed in the 1 mg/kg dose level. No adverseevents were observed following dosing, with mice receiving HER2-5demonstrating robust weight gain throughout the study (FIG. 13 ).

1. A composition comprising a plurality of drug molecules of formula I:

wherein: X is

Y is Y₁ or Y₂ further wherein Y₁ is

or H; Y₂ is —Cl, —Br, —I, or

Z is H or SO₃H; R₁ and R₂ are independently selected from H or alkyl; nis an integer from 0 to 50; and wherein the drug molecules present inthe composition comprises a mixture of at least two diastereomers, afirst diastereomer and a second diastereomer, further wherein said firstdiastereomer and second diastereomer are otherwise identical, exceptthat said first and second diastereomers have different stereochemicalconfiguration at a chiral carbon represented by (*) in formula X,wherein said chiral carbon atom is a carbon atom that is bound to asulfur atom, and said first or second diastereomer is present in adiastereomeric excess of greater than 50%.
 2. The composition of claim1, wherein n is 1, and R₁ and R₂ are each independently hydrogen.
 3. Thecomposition of claim 1, wherein the drug molecules are present in thecomposition in a diastereomeric excess of at least 95%.
 4. Thecomposition of claim 1, wherein formula I is represented by:

or mixtures thereof in a diastereomeric excess of greater than 50%.
 5. Acomposition comprising a plurality of ligand-drug conjugates of FormulaII:

wherein: A is

W is selected from S, O, or NR₃; L is a ligand; further wherein: L iscapable of binding to a cell or cell population; R₁, R₂ and R₃ are eachindependently selected from H or alkyl; n is an integer from 0 to 10; pis an integer from 1 to 10; and wherein the ligand-drug conjugates arepresent in the composition in a diastereomeric excess of greater than50%.
 6. The composition of claim 5, wherein the ligand is an antibody oran antigen-binding fragment thereof, W is NH, and R₁, R₂ are eachindependently selected from H.
 7. The composition of claim 6, whereinthe antibody or antigen-binding fragment thereof specifically binds atumor-associated antigen.
 8. The composition of claim 7, wherein theligand-drug conjugates are present in the composition in adiastereomeric excess of more than 95%.
 9. The composition of claim 7,wherein the tumor-associated antigen is selected from the groupconsisting of AFP, ALK, BAGE proteins, β-catenin, brc-abl, BRCA1, BORIS,CA9, carbonic anhydrase IX, caspase-8, CD40, CDK4, CEA, CTLA4, CLEC12A,cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML,EphA2, Fra-1, FOLR1, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH,glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT,LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1,mesothelin, ML-IAP, Muc1, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62,NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR,PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3,Steap-1, Steap-2, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TRP-1, TRP-2,tyrosinase, and uroplakin-3.
 10. A method for preparing compositioncomprising a plurality of drug molecules of formula I:

wherein X is

Y is Y₁ or Y₂ further wherein Y₁ is

or H; Y₂ is —Cl, —Br, —I, or

R₁ and R₂ are independently selected from H or alkyl; n is an integerfrom 0 to 50; and wherein the drug molecules present in the compositioncomprises a mixture of at least two diastereomers, a first diastereomerand a second diastereomer, further wherein said first diastereomer andsecond diastereomer are otherwise identical, except that said first andsecond diastereomers have different stereochemical configuration at achiral carbon represented by (*) in formula X, wherein said chiralcarbon atom is a carbon atom that is bound to a sulfur atom, and saidfirst or second diastereomer is present in a diastereomeric excess ofgreater than 50%, the method comprising: (a) providing a mixturecomprising (i) a starting material which has a formula III:

(ii) a compound of formula IV:

Y₁ is

or H; Y₂ is —Cl, —Br, —I, or

Z is H or SO₃H; R₁ and R₂ are independently selected from H or alkyl;and each n is an integer from 0 to 50; (iii) an organic solvent, (iv)water, and (v) a solid substrate; (b) allowing the mixture of step (a)to react until some of the starting material is converted to thecompound of formula I; and (c) removing crude compound of formula I fromthe mixture of step (b).
 11. The method of claim 10, further comprising(d) purifying the compound of formula I obtained in step (c).
 12. Themethod of claim 10, wherein the solid substrate is selected from thegroup consisting of silica gel, celite, alumina, a zeolite, and crushedmolecular sieves.
 13. The method of claim 10, wherein n is 1, and R₁ andR₂ are each independently hydrogen.
 14. The method of claim 10, whereinthe organic solvent comprises a polar aprotic solvent.
 15. The method ofclaim 14, wherein the polar aprotic solvent comprises acetonitrile. 16.The method of claim 10, wherein the organic solvent and the water arepresent in ratio from about 1:1 to about 4:1 or from about 1:1 to about10:1.
 17. The method of claim 10, wherein the molar ratio of thestarting material having formula III and the compound of formula IV isfrom about 1:1 to about 1:3.
 18. The method of claim 10, furthercomprising combining the compound of formula I with an antibody orantigen-binding fragment thereof to make an antibody-drug conjugate. 19.The method of claim 18, wherein the compound of formula I is attached tothe antibody or antigen-binding fragment via an S, O, or NR₃.
 20. Themethod of claim 10, wherein the formula I is represented by thefollowing structure:

in a diastereomeric excess of greater than 50%. 21.-30. (canceled)